The present invention provides fibrocyte-based vaccine formulations made from isolated fibrocytes. The present invention further provides a method for establishing an immune response against a specific antigen by administering a fibrocyte-based vaccine formulation, such as one made by pulsing fibrocytes in culture with the antigen peptide or protein, or transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes.
The immune response involves a) recognition of a specific antigen by a lymphocyte, b) elaboration of specific cellular and humoral effectors, leading to c) elimination of the antigen by specific effector cells such as T-lymphocytes and antibodies derived from B-cells, which mediate cellular and humoral immune responses, respectively. The cellular immune response begins with the recognition of antigen on the surface of an antigen-presenting cell (APC). Cellular antigen recognition is through a subset of lymphocytes called T-lymphocytes. T-lymphocytes include at least two functional subsets. These include T-helper lymphocytes (TH) that usually express the CD4 surface marker and cytotoxic T-lymphocytes (CTL) that usually express the CD8 surface maker. Both T-cell subsets express an antigen receptor that can recognize a given antigen, usually a peptide. The peptide, lipid or carbohydrate antigen needs to be associated with a major histocompatibility molecule (MHC) expressed on the surface of the APC. T-cells bearing the CD4 surface marker generally recognize antigens associated with MHC class II molecules. T-cells expressing the CD8 surface marker generally recognize antigens associated with MHC class I molecules.
In the case of protein antigens, a T-cell antigen receptor can only recognize peptides associated with MHC molecules at the surface of an APC. Thus, cellular proteins need to be processed into such peptides and transported with MHC molecules at the cell surface. This is referred to as xe2x80x9cantigen processing.xe2x80x9d Exogenous proteins, phagocytosed by the APC, are broken down into peptides that are transported on MHC class II molecules to the cell surface where they can be recognized by CD4+ cells. In contrast, endogenous proteins synthesized by the APC are also broken down into peptides, but are transported by MHC class I molecules, where they are transported to the cell surface and recognized by CD8+ T-cells.
When a T-cell binds through its antigen receptor to its cognate peptide-MHC complex on the APC, the binding generates a first signal from the T-cell membrane towards its nucleus. However, this first signal is insufficient to activate the T-cell, at least as measured by induction of IL-2 synthesis and secretion. T-cell activation only occurs if a second signal or co-stimulatory signal is generated by the binding of other APC molecules to their appropriate receptors on the T-cell surface. The best known co-stimulatory molecules identified to date on APC are B7-1 and B7-2; both bind CTLA4/CD28 counter receptor on T-lymphocytes. In addition, adhesion molecules CD11a, CD54 and CD58 are thought to be important as co-stimulatory molecules. The ability to present peptide antigens together with co-stimulatory molecules in such a way as to activate T-cells is hereafter referred to as antigen presentation. Only APC""s have the capacity to present antigens to CD4+(predominantly TH) and CD8+(predominantly CTL) T-cells, leading to the development of humoral and cellular immune responses.
Fibrocytes
APCs are heterogeneous in their cell lineage and functional properties. They include distinct cell types such as B-lymphocytes, T-lymphocytes, monocytes and macrophages, Langerhan cells and dendritic cells. A new isolated population of blood-borne fibroblast-like cells that rapidly enter sites of tissue injury, synthesize connective tissue matrix, and express fibrogenic cytokines (Bucala et al., Mol. Medicine 1:71-81, 1994) have been described. Termed xe2x80x9cfibrocytesxe2x80x9d, these isolated cells can proliferate in culture, are adherent to culture plates, and display a spindle-shaped morphology. Fibrocytes have a distinct phenotype, characterized by surface markers, and synthesize the fibroblast products collagen I, collagen III and fibronectin and express several leukocyte-associated cell surface antigens, including CD45RO and the hematopoietic stem cell antigen CD34. Fibrocytes do not express a variety of endothelial, epithliial or smooth-muscle markers and are negative for non-specific esterases as well as the monocyte/macrophage specific markers CD4 and CD 16 (Bucala et al., 1994 infra.). Fibrocytes do not express CD25, a protein expressed constitutively by dendritic cells (Freudenthal and Steinman Proc. Natl. Acad. Sci. USA 87:7698-7702, 1990) or CD1a, a Langerhans cell marker (Chu and Jaffe, Br. J Cancer Suppl. 23:S4-S10, 1994). Thus, an isolated fibrocyte cell is a unique cell type with a defined phenotype readily distinguishable from other known APC types.
Fibrocytes expresses the cell surface molecules that are required for antigen presentation and are potent inducers of antigen-specific T-cell proliferation in vitro and in vivo. Isolated fibrocytes express high levels of class II MHC molecule HLA-DR. For example, mouse isolated fibrocytes, pulsed in vitro with foreign antigen and injected into the skin, were found to migrate to regional lymph notes and to prime T-cells. An example is shown pulsing fibrocytes with protein p120, both proteins expressed of HIV.
Immunohistochemical analyses of tissues undergoing fibrosis and tissue remodeling have identified fibrocytes to be present within areas of extracellular matrix deposition, providing direct evidence for the participation of fibrocytes in the host repair response to tissue injury (Bucala et al., 1994 infra.). Peripheral blood fibrocytes express a full complement of surface proteins required for antigen presentation, are potent stimulators of antigen-specific T cells in vitro, and migrate to lymph nodes and sensitize naive T cells in situ.
Fibrocytes also have been shown to secrete a number of inflammatory cytokines in vitro and are a particularly abundant source of the potent CD4+ T cell chemoattractants, macrophage inflammatory proteins (MIP-1xcex1 and 1xcex2) (Schall et al., J. Exp. Med. 177:1821-1825, 1993). The entry of CD4+ T cells into areas of tissue damage is considered to be an essential requirement for the generation of an antigen-specific immune response (Wahl and Wahl. in Wound Healing: Biochemical and Clinical Aspects, eds. Cohen et al. eds. Saunders Company, Philadelphia, 40-62, 1992). Fibrocytes thus may act to not only activate but to also recruit CD4+ T cells into the tissue repair micro-environment.
The constitutive expression by fibrocytes of the surface proteins known to be necessary for antigen presentation contrasts with what has been described for tissue fibroblasts which require activation by inteferon-xcex3 to express measurable quantities of HLA-DR (Geppert and Lipsky, J. Immunol. 135:3750-3762, 1985). Although several tissue-derived cells have been shown to be capable of presenting antigen to memory T cells, including dermal fibroblasts, endothelial cells, and melanocytes (Pober et al., J. Exp. Med., 157:1339-1353, 1983; and Le Poole et al., J. Immunol., 151:12,7284-7292, 1993), sensitization of native T cells has been considered to be a particular function of dendritic cells (Inaba et al., J. Exp. Med. 172:631-640, 1990; and Levin et al., J. Immunol. 151:12,6742-6750, 1993). Fibrocytes also preset antigen to naive T cells but are distinct from dendritic cells and their precursors not only in their growth properties (fibrocytes are an adherent, proliferating cell population whereas dendritic cells are non-adhering and poorly proliferating) but also in their surface protein expression (collagen+/CD13+/CD34+/CD25xe2x88x92/CD10xe2x88x92/CD38xe2x88x92).
The present invention provides fibrocyte-based vaccine formulations made from isolated fibrocytes and an antigenic component, wherein the antigenic component is selected from the group consisting of pulsed antigen (protein, peptide, lipid, carbohydrate or a synthetic compound), a gene encoding specific antigenic determinants of proteins or peptides, tumor cells, and membrane fragments from tumor cells. Preferably, the antigenic component is a tumor cell or a tumor cell membrane that is fused with the isolated fibrocytes to form a fused cell that is the fibrocyte-based vaccine formulation. Preferably, the fusion process occurs ex vivo and the fibrocyte-based vaccine formulation is administered in vivo. Preferably the fibrocyte-based vaccine formulation is directed against an infectious disease and is formed by transfecting fibrocytes with a gene encoding a viral or a bacterial antigenic determinant that is displayed as an MHC class II antigenic determinant on the surface of the transfected fibrocytes.
The present invention further provides a method for establishing an immune response against a specific antigen by administering a fibrocyte-based vaccine formulation, such as one made by pulsing fibrocytes in culture with the antigen peptide or protein, or transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes. Preferably, the method provides an immune response is directed against a tumor antigen, a viral antigen, or a bacterial antigen. Preferably, the fibrocyte-based vaccine is produced by a process comprising pulsing fibrocytes in culture with an antigen peptide or protein, or transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins, or by fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes.
The present invention further provides a process for producing a fibrocyte-based vaccine formulation, comprising: (a) obtaining isolated fibrocytes, and (b) either (i) pulsing fibrocytes in culture with an antigen peptide or protein; or (ii) transfecting fibrocytes with genes encoding specific antigenic determinants of peptides or proteins; or (iii) fusing tumor cells (whole cells or membrane fragments thereof) with fibrocytes. Preferably, the fusion process (iii) is performed by mixing a population of isolated fibrocyte cells taken from a patient having cancer with tumor cells taken from the patient in a fusion catalyst, and isolating fused cells from non-fused fibrocytes or tumor cells by density gradient means to form a fibrocyte-based vaccine formulation. Most preferably, the process further comprises irradiating the isolated fibrocyte-based vaccine formulation to insure that it is incapable of growth after in vivo administration.